title: Analysis of the reconstitution of oligomeric enzymes by cross-linking with glutaraldehyde: kinetics of reassociation of lactic dehydrogenase.
abstract: Cross-linking with glutaraldehyde with subsequent NaDodSO4-polyacrylamide gel electrophoresis has been introduced as a convenient method for studying the association of oligomeric proteins [Hermann, R., Rudolph, R., & Jaenicke, R. (1979) Nature (London) 277, 243-245]. In the present paper, an improved version of this approach was applied to the analysis of the complex association behavior of the tetrameric lactic dehydrogenase from pig muscle. Monomers, dimers (as intermediates of reconstitution), and tetramers could be quantitatively determined during reconstitution. The initial fast formation of dimers from monomers does not reach completion; a certain amount of monomers remains during the whole reconstitution process. Monomers and dimers disappear parallel to the formation of tetramers. The reassociation behavior of lactic dehydrogenase is described by a kinetic mechanism comprising a dissociation-association equilibrium of monomers and dimers [characterized by an equilibrium constant K = (3 +/- 1) X 10(8) L mol-1] followed by the rate-limiting association of dimers to tetramers [described by a second-order rate constant k = (3.15 +/- 0.15) X 19=0(4) L mol-1 s-1]. Tetramerization is found to strictly parallel reactivation.
mesh_terms: D000447:Aldehydes; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005976:Glutaral; D007700:Kinetics; D007770:L-Lactate Dehydrogenase; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D013552:Swine
chemical_list: D000447:Aldehydes; D003432:Cross-Linking Reagents; D007770:L-Lactate Dehydrogenase; D005976:Glutaral